Artificially induced tetraploid masu salmon have the ability to form primordial germ cells

Diploid gametes generated with tetraploid animals are a stepping stone to improving chromosome manipulation techniques. However, artificially induced tetraploid individuals generally die soon after hatching. Diploid gametes could be induced by in vivo cultures of tetraploid primordial germ cells (PGCs) through germ-line chimera. In the present study, characteristics of PGCs were studied in inviable tetraploid masu salmon, Oncorhynchus masou. Histological observation of tetraploid embryos revealed that the same or smaller numbers of PGCs were observed and they migrate into the genital ridges as did diploid PGCs during gonadogenesis. By whole-mount in situ hybridization using vasa messenger RNA (mRNA), 4–35 vasa-positive signals were detected in a pair of genital ridges of tetraploids. By cytological observation of genital ridge cell suspensions, several large round cells were observed, some of which extended pseudopodia. They also contained large nuclei and round granules in their cytoplasm, characteristics of PGCs. As the results suggest that inviable artificial tetraploids have PGCs, we expect to achieve diploid gamete production through surrogate propagation and tetraploid fish production.     

Effect of calcium ion on the thermal denaturation of subfragment-1 and rod regions of squid myosin upon the heating of myofibrils

Thermal inactivation of Ca²⁺ ATPase of squid myofibrils was significantly suppressed in the presence of Ca²⁺. Monomeric myosin content decreased much faster than Ca²⁺ ATPase inactivation in Ca medium, which was well explained by fast rod denaturation. In contrast, rod denaturation was slower than S-1 in EDTA medium. The decrease in monomeric myosin content was explained by faster S-1 denaturation. Comparing the S-1 and rod denaturation rates at a fixed temperature, it was concluded that S-1 denaturation was suppressed by Ca²⁺ whereas the rod denaturation was not. An unfolding experiment with isolated myosin rod confirmed that there was no stabilizing effect of Ca²⁺ on myosin rod. It was concluded that significant stabilization of the S-1 portion by Ca²⁺ generated the apparently different myosin denaturation patterns in the two media.     

Relative potencies of natural estrogens on vitellogenin and choriogenin levels in the Indian freshwater spotted snakehead, <Emphasis Type="Italic">Channa punctata</Emphasis>: in vivo and in vitro studies

The relative efficacies of three natural estrogens viz., estrone (E₁), estradiol-17β (E₂) and estriol (E₃) to induce synthesis of vitellogenin (Vg) and choriogenin (Chg) were assessed in primary hepatocyte cultures of the Indian freshwater spotted snakehead, Channa punctata. Hepatocytes were isolated from the spotted snakehead liver by a non-enzymatic protocol. Optimum culture conditions were standardized for ensuring their viability and functioning. Isolated hepatocytes were cultured for 48 h for monolayer formation and then exposed to various concentrations (0.001–10 μM) of the three estrogens. Competitive homologous ELISAs, developed and validated for spotted snakehead Vg and Chg were employed to determine the amounts of these two proteins secreted into the culture medium after 48 h of incubation. The results reveal that although all the three estrogens were effective in inducing the production of Vg and Chg in a dose-dependent manner, there were differences in their relative potencies. Of three estrogens, E₁ was the least potent and could induce synthesis of Vg and Chg only at a minimum concentration of 0.5 μM; whereas significant levels of both the proteins were quantified in culture medium by exposing the hepatocytes to E₂ or E₃ even at a concentration of 0.001 μM. All three estrogens were effective in inducing synthesis of Vg and Chg in vivo also. These results suggest the possibility of employing the above in vitro experimental design to monitor the presence of estrogens/estrogen-like chemicals in natural waters, which could interfere with the estrogen receptor system of fish. This study further points to the possibility of using Chg, in addition to Vg, as a parameter for screening various chemicals for their estrogenic activity.     

Effect of in vitro exposure to Vibrio vulnificus on hydroelectrolytic transport and structural changes of sea bream (Sparus aurata L.) intestine

The everted gut sac technique has been used to investigate the effect of Vibrio vulnificus on water and electrolyte (Na⁺, K⁺, Cl⁻, HCO₃ ⁻) transport on the intestine of sea bream (Sparus aurata L.). Both the anterior and the posterior intestine were incubated in a medium containing 10⁸ V. vulnificus cells ml⁻¹ at 25°C for 2 h. The presence of V. vulnificus resulted in a significant reduction (P < 0.05) of water absorption in the anterior intestine, while sodium absorption in the anterior (P < 0.01) and posterior (P < 0.05) intestine was elevated. Chloride absorption was increased, but the changed was not significant, while potassium absorption decreased significantly (P < 0.05), but only in the posterior intestine. Incubation the sea bream intestine with V. vulnificus did not affect carbonate secretion in the anterior segment, whereas high secretion was stimulated in the posterior segment (P < 0.01). Histological evaluations demonstrated damage in the anterior intestine of sea bream that was characterized by the detachment of degenerative enterocytes, alterations in the microvilli, and the presence of a heterogenous cell population, indicating inflammation. Based on our results, we conclude that V. vulnificus caused cell damage to the intestine of sea bream and that the anterior intestine is more susceptible than the posterior part of the intestine. Several hypotheses are suggested to explain our observations, such as the presence of higher numbers of villosities in the anterior intestine than in the posterior one and/or the presence of endogenous bacteria in the posterior intestine which may have a protector role.     

Microsatellite marker isolation and cultured strain identification in <Emphasis Type="Italic">Carassius auratus gibelio</Emphasis>

Microsatellites have become the preferred molecular markers for strain selection and genetic breeding in fish. In this study a total of 105 microsatellites were isolated and identified in gibel carp (Carassius auratus gibelio) by microsatellite sequence searches in GenBank and other databases and by screening and sequencing of positive clones from the genomic library enriched for AG and GATA repeats. Moreover, nineteen microsatellites were randomly selected to design locus-specific primer pairs, and these were successfully used to identify and discriminate different cultured strains of gibel carp including strains A, D, L, and F. Three different types of microsatellite pattern were distinguished by the number and length of fragments amplified from the 19 primer pairs, and some microsatellite primer pairs were found to produce different microsatellite patterns among strains and strain-specific fragments. In addition, some duplicated alleles were also detected in two microsatellite patterns. Therefore, the current study provides direct molecular markers to discriminate among different cultured strains for selective breeding and aquaculture practice of gibel carp.     

Effects of light intensity on molting,growth, precocity,digestive enzyme activity,and chemical composition of juvenile Chinese mitten crab <Emphasis Type="Italic">Eriocheir sinensis</Emphasis>

A 74-day growth trial was carried out to investigate the effects of light intensity on the juvenile Chinese mitten crab, Eriocheir sinensis, under semi-natural conditions. The experiment had three light intensity treatment groups, natural light (NL), middle light (ML), and low light (LL), as light intensity became weaker. The results indicated that light intensity had no significant effect on molting interval and molting frequency but did have a significant effect on the molting weight gain of the crab. Molting weight gain in group NL was significantly higher than that in group LL (P < 0.05). Specific growth rate in weight (SGRw) and in carapace width (SGRcw), weight gain, and final body weight were significantly affected by light intensity (P < 0.05). No significant difference was detected in survival between groups (P > 0.05). The precocious rate in groups NL, ML, and LL was 26.14, 15.48, and 17.14%, respectively. The precocious rate in group NL was significantly higher than that in groups ML and LL (P < 0.05). Chemical composition of the crab body was significantly affected by light intensity, but the activity of alkaline phosphatase, trypsin, and pancreatic lipase was not significantly affected by light intensity. The results indicated that the submitted light intensity was useful in reducing the precocious rate without affecting the normal growth of juvenile E. sinensis.     

Sex reversal and growth performance in juvenile females of the freshwater crayfish <Emphasis Type="Italic">Cherax quadricarinatus</Emphasis> (Parastacidae): effect of increasing temperature and androgenic gland extract in the diet

Monosex male culture of C. quadricarinatus is proposed for increasing yields because males reach a larger size at harvest than females. An experiment was done to evaluate the effects of androgenic gland extract and temperature on sex reversal and somatic growth in early juvenile females. Females were exposed to the following experimental conditions: C: control food and temperature maintained at 26 ± 1°C; HT (high temperature): control food and temperature maintained at 28.5 ± 1°C; VHT (very high temperature): control food and temperature maintained at 31 ± 1°C; AG: food with enriched with androgenic gland (1/10 dose for each juvenile per day) and temperature maintained at (26 ± 1)°C. The juveniles were weighed, sexed, and growth increment (GI) and growth rate (GR) were calculated twice a month. The HT and AG groups differed from the C group in GI and GR indicating a greater somatic growth. At the end of the experiment, the HT and AG groups had similar weight but only the HT group had enhanced oocyte diameter, with some vitellogenic oocytes compared to the C group. In the HT group, there was significant sex reversal, as indicated by development of male and intersex male characteristics in different individuals. Both temperature and AG diet have a high potential for culture of this species because of their enhancement of somatic growth; higher temperature increases the proportion of males, a desirable characteristic of cultured populations.     

Ontogenetic development of digestive enzymes and effect of starvation in miiuy croaker <Emphasis Type="Italic">Miichthys miiuy</Emphasis> larvae

The ontogenetic development of the digestive enzymes amylase, lipase, trypsin, and alkaline phosphatase and the effect of starvation in miiuy croaker Miichthys miiuy larvae were studied. The activities of these enzymes were detected prior to exogenous feeding, but their developmental patterns differed remarkably. Trypsin activity continuously increased from 2 days after hatching (dah), peaked on 20 dah, and decreased to 25 dah at weaning. Alkaline phosphatase activity oscillated at low levels within a small range after the first feeding on 3 dah. In contrast, amylase and lipase activities followed the general developmental pattern that has been characterized in fish larvae, with a succession of increases or decreases. Amylase, lipase, and trypsin activities generally started to increase or decrease at transitions from endogenous to exogenous feeding or diet changes, suggesting that these enzymatic activities can be modulated by feeding modes. The activities of all the enzymes remained stable from 25 dah onwards, coinciding with the formation of gastric glands and pyloric caecum. These results imply that specific activities of these enzymes underwent changes due to morphological and physiological modifications or diet shift during larval development but that they became stable after the development of the digestive organs and associated glands was fully completed and the organs/glands functioned. Trypsin and alkaline phosphatase were more sensitive to starvation than amylase and lipase because delayed feeding up to 2 days after mouth opening was able to adversely affect their activities. Enzyme activities did not significantly differ among feeding groups during endogenous feeding; however, all activities were remarkably reduced when delayed feeding was within 3 days after mouth opening. Initiation of larvae feeding should occur within 2 days after mouth opening so that good growth and survival can be obtained in the culture.     

Can fermented soybean meal and squid by‐product blend be used as fishmeal replacements for Japanese flounder (Paralichthys olivaceus)?

An experiment was carried out to evaluate fermented soybean meal and squid by‐product blend (1:1) (FP) as replacement of fishmeal (FM) for Japanese flounder, Paralichthys olivaceus. Five isocaloric (19 kJ g^?1) diets were prepared by replacing 0 (FP0), 12 (FP12), 24 (FP24), 36 (FP36) and 48% (FP48) FM protein with FP. Triplicate groups of juveniles (mean weight of 3.9 g) were delivered the test diets for 8 weeks in a flow‐through sea water system. The results showed that there were no significant differences (P > 0.05) among the growth rates of fish fed FP0, FP12, FP24 and FP36 diets. Growth and nutrient utilization parameters were significantly reduced in fish fed FP48 diet. Although, whole body proximate composition of fish were not significantly affected by the dietary treatments compared to the control; methionine and phenylalanine contents were significantly decreased in FP48 group. Protein retention was also significantly decreased in the similar group of fish. Dietary treatments did not alter most of the plasma metabolites, while some of the health parameters were improved in the replacement groups. Results suggested that FP is a potential candidate for alternative protein ingredient in aquafeed and can replace 36% FM protein in the diet of Japanese flounder.